Advancing brain barriers RNA sequencing: guidelines from experimental design to publication

Abstract Background RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers re...

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Main Authors: David M. F. Francisco, Luca Marchetti, Sabela Rodríguez-Lorenzo, Eduardo Frías-Anaya, Ricardo M. Figueiredo, BtRAIN Network, Peter Winter, Ignacio Andres Romero, Helga E. de Vries, Britta Engelhardt, Rémy Bruggmann
Format: Article
Language:English
Published: BMC 2020-08-01
Series:Fluids and Barriers of the CNS
Online Access:http://link.springer.com/article/10.1186/s12987-020-00207-2
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spelling doaj-5a307900bb7c46628a368ed0b70f18fc2020-11-25T03:46:33ZengBMCFluids and Barriers of the CNS2045-81182020-08-0117113310.1186/s12987-020-00207-2Advancing brain barriers RNA sequencing: guidelines from experimental design to publicationDavid M. F. Francisco0Luca Marchetti1Sabela Rodríguez-Lorenzo2Eduardo Frías-Anaya3Ricardo M. Figueiredo4BtRAIN NetworkPeter Winter5Ignacio Andres Romero6Helga E. de Vries7Britta Engelhardt8Rémy Bruggmann9Interfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of BernTheodor Kocher Institute, University of BernMS Center Amsterdam, Amsterdam Neuroscience, Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit AmsterdamSchool of Life, Health and Chemical Sciences, The Open UniversityGenXPro GmbHGenXPro GmbHSchool of Life, Health and Chemical Sciences, The Open UniversityMS Center Amsterdam, Amsterdam Neuroscience, Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit AmsterdamTheodor Kocher Institute, University of BernInterfaculty Bioinformatics Unit and Swiss Institute of Bioinformatics, University of BernAbstract Background RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process. Main body In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN ( https://www.btrain-2020.eu/ ) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community. Conclusion Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community.http://link.springer.com/article/10.1186/s12987-020-00207-2
collection DOAJ
language English
format Article
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author David M. F. Francisco
Luca Marchetti
Sabela Rodríguez-Lorenzo
Eduardo Frías-Anaya
Ricardo M. Figueiredo
BtRAIN Network
Peter Winter
Ignacio Andres Romero
Helga E. de Vries
Britta Engelhardt
Rémy Bruggmann
spellingShingle David M. F. Francisco
Luca Marchetti
Sabela Rodríguez-Lorenzo
Eduardo Frías-Anaya
Ricardo M. Figueiredo
BtRAIN Network
Peter Winter
Ignacio Andres Romero
Helga E. de Vries
Britta Engelhardt
Rémy Bruggmann
Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
Fluids and Barriers of the CNS
author_facet David M. F. Francisco
Luca Marchetti
Sabela Rodríguez-Lorenzo
Eduardo Frías-Anaya
Ricardo M. Figueiredo
BtRAIN Network
Peter Winter
Ignacio Andres Romero
Helga E. de Vries
Britta Engelhardt
Rémy Bruggmann
author_sort David M. F. Francisco
title Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
title_short Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
title_full Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
title_fullStr Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
title_full_unstemmed Advancing brain barriers RNA sequencing: guidelines from experimental design to publication
title_sort advancing brain barriers rna sequencing: guidelines from experimental design to publication
publisher BMC
series Fluids and Barriers of the CNS
issn 2045-8118
publishDate 2020-08-01
description Abstract Background RNA sequencing (RNA-Seq) in its varied forms has become an indispensable tool for analyzing differential gene expression and thus characterization of specific tissues. Aiming to understand the brain barriers genetic signature, RNA seq has also been introduced in brain barriers research. This has led to availability of both, bulk and single-cell RNA-Seq datasets over the last few years. If appropriately performed, the RNA-Seq studies provide powerful datasets that allow for significant deepening of knowledge on the molecular mechanisms that establish the brain barriers. However, RNA-Seq studies comprise complex workflows that require to consider many options and variables before, during and after the proper sequencing process. Main body In the current manuscript, we build on the interdisciplinary experience of the European PhD Training Network BtRAIN ( https://www.btrain-2020.eu/ ) where bioinformaticians and brain barriers researchers collaborated to analyze and establish RNA-Seq datasets on vertebrate brain barriers. The obstacles BtRAIN has identified in this process have been integrated into the present manuscript. It provides guidelines along the entire workflow of brain barriers RNA-Seq studies starting from the overall experimental design to interpretation of results. Focusing on the vertebrate endothelial blood–brain barrier (BBB) and epithelial blood-cerebrospinal-fluid barrier (BCSFB) of the choroid plexus, we provide a step-by-step description of the workflow, highlighting the decisions to be made at each step of the workflow and explaining the strengths and weaknesses of individual choices made. Finally, we propose recommendations for accurate data interpretation and on the information to be included into a publication to ensure appropriate accessibility of the data and reproducibility of the observations by the scientific community. Conclusion Next generation transcriptomic profiling of the brain barriers provides a novel resource for understanding the development, function and pathology of these barrier cells, which is essential for understanding CNS homeostasis and disease. Continuous advancement and sophistication of RNA-Seq will require interdisciplinary approaches between brain barrier researchers and bioinformaticians as successfully performed in BtRAIN. The present guidelines are built on the BtRAIN interdisciplinary experience and aim to facilitate collaboration of brain barriers researchers with bioinformaticians to advance RNA-Seq study design in the brain barriers community.
url http://link.springer.com/article/10.1186/s12987-020-00207-2
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